RESUMEN
Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-ß-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.
Asunto(s)
Fijación del Nitrógeno , Phaseolus/microbiología , Rhizobium/clasificación , Rhizobium/metabolismo , Sinorhizobium/clasificación , Sinorhizobium/metabolismo , Simbiosis , Datos de Secuencia Molecular , Oxidorreductasas/genética , Rhizobium/aislamiento & purificación , Rhizobium/fisiología , Análisis de Secuencia de ADN , Sinorhizobium/genética , Sinorhizobium/aislamiento & purificaciónRESUMEN
Rhizobia are Gram-negative bacteria that live in soils and associate with leguminous plants to establish nitrogen-fixing symbioses. The ability of these bacteria to undergo horizontal gene transfer (HGT) is thought to be one of the main features to explain both the origin of their symbiotic life-style and the plasticity and dynamics of their genomes. In our laboratory we have previously characterized at the species level the non-pSym plasmid mobilome in Sinorhizobium meliloti, the symbiont of Medicago spp., and have found a high incidence of conjugal activity in many plasmids (Pistorio et al., 2008). In this work we characterized the Dtr (DNA-transfer-and-replication) region of one of those plasmids, pSmeLPU88b. This mobilization region was found to represent a previously unclassified Dtr type in rhizobia (hereafter type-IV), highly ubiquitous in S. meliloti and found in other genera of Gram-negative bacteria as well; including Agrobacterium, Ochrobactrum, and Chelativorans. The oriT of the type-IV Dtr described here could be located by function within a DNA fragment of 278 bp, between the divergent genes parA and mobC. The phylogenetic analysis of the cognate relaxase MobZ indicated that this protein groups close to the previously defined MOB(P3) and MOB(P4) type of enzymes, but is located in a separate and novel cluster that we have designated MOB(P0). Noteworthy, MOB(P0) and MOB(P4) relaxases were frequently associated with plasmids present in rhizospheric soil bacteria. A comparison of the nod-gene locations with the phylogenetic topology of the rhizobial relaxases revealed that the symbiotic genes are found on diverse plasmids bearing any of the four Dtr types, thus indicating that pSym plasmids are not specifically associated with any particular mobilization system. Finally, we demonstrated that the type-IV Dtr promoted the mobilization of plasmids from S. meliloti to Sinorhizobium medicae as well as from these rhizobia to other bacteria by means of their own helper functions. The results present an as-yet-unclassified and seemingly ubiquitous conjugal system that provides a mechanistic support for the HGT between sympatric rhizobia of Medicago roots, and between other soil and rhizospheric bacteria.
Asunto(s)
ADN Bacteriano/aislamiento & purificación , Genes Bacterianos , Bacterias Gramnegativas/genética , Sinorhizobium meliloti/genética , Sinorhizobium/genética , Microbiología del Suelo , Secuencia de Bases , Conjugación Genética , ADN Bacteriano/genética , Transferencia de Gen Horizontal , Bacterias Gramnegativas/clasificación , Medicago/microbiología , Datos de Secuencia Molecular , Fijación del Nitrógeno , Filogenia , Raíces de Plantas/microbiología , Plásmidos , Sinorhizobium/clasificación , Sinorhizobium meliloti/clasificación , Simbiosis/genética , SimpatríaRESUMEN
Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum. A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA-DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima (Rhizobium, Sinorhizobium and Mesorhizobium) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella.
Asunto(s)
Fabaceae/microbiología , Fijación del Nitrógeno , Rhizobium , Simbiosis , Técnicas de Tipificación Bacteriana , Fabaceae/crecimiento & desarrollo , México , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Filogenia , Rhizobium/clasificación , Rhizobium/genética , Rhizobium/fisiología , Análisis de Secuencia de ADN , Sinorhizobium/clasificación , Sinorhizobium/genética , Sinorhizobium/fisiología , Especificidad de la Especie , Simbiosis/genéticaRESUMEN
A new lineage of Ensifer nodulating the American legume Acacia angustissima in the tropical forest of Chiapas and Morelos, Mexico is described. Bacteria were identified as Ensifer with ssb or nolR specific primers. Phylogenetic analysis with partial sequences of the five chromosomal genes gyrA, nolR, recA, rpoB and rrs revealed that this new lineage is related to African Ensifer terangae. The results of total DNA-DNA hybridization and selected phenotypic tests among the A. angustissima strains and E. terangae indicated that they belong to different species. The phylogeny with the symbiotic nifH gene also separates this group as a different clade but with close affinities to bacteria belonging to the genus Ensifer isolated from American hosts. ITTG R7(T) (=CFN ER1001, HAMBI 2910, CIP 109033, ATCC BAA-1312, DSM18446) is the type strain of a new species for which the name Ensifer mexicanus sp. nov. is proposed.
Asunto(s)
Acacia/microbiología , Alphaproteobacteria/clasificación , Alphaproteobacteria/genética , Alphaproteobacteria/aislamiento & purificación , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Genes Bacterianos/genética , México , Fijación del Nitrógeno/genética , Hibridación de Ácido Nucleico , Filogenia , Homología de Secuencia , Sinorhizobium/clasificación , Especificidad de la Especie , SimbiosisRESUMEN
The sinorhizobia isolated from root nodules of Acacia species native of Mexico constitute a diverse group of bacteria on the basis of their metabolic enzyme electromorphs but share restriction patterns of the PCR products of 16S rRNA genes and a common 500 kb symbiotic plasmid. They are distinguished from other Sinorhizobium species by their levels of DNA-DNA hybridization and the sequence of 16S rRNA and nifH genes. nolR gene hybridization patterns were found useful to identify sinorhizobia and characterize species. A new species, Sinorhizobium americanus, is described and the type strain is CFNEI 156 from Acacia acatlensis.
Asunto(s)
Acacia/microbiología , Fijación del Nitrógeno , Sinorhizobium/clasificación , Acacia/clasificación , Secuencia de Bases , Farmacorresistencia Bacteriana Múltiple , Enzimas/análisis , Enzimas/clasificación , Enzimas/metabolismo , Kanamicina/farmacología , México , Ácido Nalidíxico/farmacología , Hibridación de Ácido Nucleico , Filogenia , Raíces de Plantas/microbiología , ARN Ribosómico 16S/genética , Sinorhizobium/genética , Sinorhizobium/aislamiento & purificación , Sinorhizobium/ultraestructura , Microbiología del SueloRESUMEN
Sinorhizobium morelense sp. nov. is described to designate a group of bacteria isolated from root nodules of Leucaena leucocephala. S. morelense shows 98% 16S rRNA gene sequence similarity to some Sinorhizobium species and to Ensifer adhaerens. This novel species is distinguished from other Sinorhizobium species and from E. adhaerens by DNA-DNA hybridization, 165 rRNA gene restriction fragments and sequence and some distinctive phenotypic features. Strains of this species are highly resistant to some antibiotics, such as carbenicillin (1 mg ml(-1)), kanamycin (500 microg ml(-1)) and erythromycin (300 microg ml(-1)). They do not form nodules, but a nodulating strain, Lc57, is closely related to the novel species. Strain Lc04T (= LMG 21331T = CFN E1007T) is designated as the type strain of this novel species.